First Natural Endocranial Cast of a Fossil Snake (Cretaceous of Patagonia, Argentina) LAURA N. TRIVI ~NO ,1* ADRIANA M. ALBINO,2 MAR�IA T. DOZO,3 AND JORGE D. WILLIAMS1 1CONICET, Secci�on Herpetolog�ıa, Museo de La Plata, Universidad Nacional de La Plata, Paseo del Bosque s/n, La Plata B1900FWA, Argentina 2CONICET, Departamento de Biolog�ıa, Universidad Nacional de Mar del Plata, Funes 3250, Mar del Plata B7602AYJ, Argentina 3CONICET, Centro Nacional Patag�onico, Instituto Patag�onico de Geolog�ıa y Paleontolog�ıa, Boulevard Brown 2915, Puerto, Madryn U9120ACD, Argentina ABSTRACT In this study, we describe a natural endocranial cast included in a partially preserved medium-sized skull of the Upper Cretaceous South American snake Dinilysia patagonica. The endocast is composed of sedi- mentary filling of the cranial cavity in which the posterior brain, the ves- sels, the cranial nerves, and the inner ear surrounded by delicate semicircular canals, are represented. It is simple in form, with little dif- ferentiation between the three main areas (Forebrain, Midbrain, and Hindbrain), and without flexures. The nervous system is well preserved. The posterior brain surface is smooth, except for two small prominences that make up the cerebellum. A large inner ear is preserved on the right side; it consists of a voluminous central mass, the vestibule, which occu- pies most of the space defined by the three semicircular canals. In partic- ular, the lateral semicircular canal is very close to the vestibule. This characteristic, in combination with the medium to large body size of Dini- lysia, its large skull and dorsally exposed orbits, and vertebrae bearing a rather high neural spine on a depressed neural arch, suggests that this snake would have had a semifossorial lifestyle. Anat Rec, 301:9–20, 2018. VC 2017 Wiley Periodicals, Inc. Key words: snakes; Cretaceous; Dinilysia patagonica; palaeoneurology INTRODUCTION Although for a long time Palaeoneurology has been based exclusively on natural or artificial endocasts (Jerison, 1973; Edinger, 1975; Hopson, 1979), the use of high resolution X-ray computed tomography has increased the number of artificial endocranial casts of the nervous system in a wide range of extinct vertebrates, especially mammals and archo- saurs (Witmer et al., 2008 and references cited there). Stud- ies on Palaeoneurology in snakes were nonexistent (Hopson, 1979), but Yi (2013, 2015) and Yi and Norell (2015) recently analyzed some aspects of the inner ear anatomy in a variety of snakes through X-ray computed tomography which builds three-dimensional models that are virtual endocasts of the bony inner ear labyrinth. The materials analyzed by these authors included a skull of the extinct snake Dinilysia pata- gonica (MACN-RN 1014). *Correspondence to: Laura N. Trivi~no, CONICET, Secci�on Herpetolog�ıa, Museo de La Plata, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900FWA, La Plata, Argentina. E-mail: lauratrivinio@fcnym.unlp.edu.ar Received 13 May 2016; Revised 20 March 2017; Accepted 23 March 2017. DOI 10.1002/ar.23686 Published online 16 September 2017 in Wiley Online Library (wileyonlinelibrary.com). THE ANATOMICAL RECORD 301:9–20 (2018) VVC 2017 WILEY PERIODICALS, INC. http://orcid.org/0000-0003-0969-569X Dinilysia patagonica is an Upper Cretaceous snake from Argentina, known for exceptionally well preserved cranial and postcranial remains recovered in northwest Patagonia (Smith-Woodward, 1901; Estes et al., 1970; Hecht, 1982; Rage and Albino, 1989; Albino and Cald- well, 2003; Albino, 2007; Scanferla and Canale, 2007; Caldwell and Calvo, 2008; Filippi and Garrido, 2012; Zaher and Scanferla, 2012; Trivi~no and Albino, 2015). Taking into account the osteological anatomy, Dinilysia patagonica is considered basal in most ophidian clado- grams (Caldwell, 1999; Rieppel and Zaher, 2000; Scanlon and Lee, 2000; Tchernov et al., 2000; Lee and Scanlon; 2002; Zaher and Scanferla, 2012; Hsiang et al., 2015); thus, relevant neuroanatomical data of this species could add substantial information about the early evolution of snakes. In this paper, a natural cranial endocast of an extinct snake is described for first time. The material corresponds to a fragmentary skull of Dinilysia pata- gonica (MLP 79-II-27-1) previously reported by Cald- well and Albino (2002) and Albino (2007). This skull includes an endocast preserved by sedimentary filling of the cranial cavity in which the posterior brain, the vessels, the cranial nerves, the inner ear and the semi- circular canals are preserved (Figs. 1 and 2). Both the osteology and the soft tissue cast of this unpublished skull are also described herein. The results of this study are compared with the conclusions published by Yi (2013, 2015) and Yi and Norell (2015) in order to test the hypothesis about the burrowing origin of mod- ern snakes. MATERIALS AND METHODS What exactly does a natural endocranial cast repre- sent? The nervous system of reptiles is tubular, linear in organization, and has some degree of dorsoventral flex- ure along its length (Wyneken, 2007). The brain cavity is limited by a tubular cranium composed rostrally by the cartilaginous ethmoids, laterally by the bony otic series, ventrally by the basisphenoid and laterosphe- noids, and caudally by the occipital series (Wyneken, 2007). The tubular cranium is covered by the supraocci- pital, parietal, and frontal bones, and there is a subdural space (below the dura mater) and an epidural space (above the dura mater). An endocast is the sedimentary infill of a cavity that forms a three-dimensional struc- ture. Therefore, what it is known as a natural cranial endocast is the filling of the intracranial cavity that con- tains the brain with its cranial nerves, the meninges that cover and protect them, and the blood vessels. These casts may provide approximations of the brain morphology, with the possibility to see details of some superficial structures (Macrini et al., 2006). In extant reptiles, the volume of the brain does not determine the topographic relationships between brain and skull (Starck, 1979). The size of the brain is deter- mined by the body size whereas the volumes of individual cerebral segments are dependent on the development of sense organs (Starck, 1979). In most reptiles, the brain is smaller than the intracranial cavity. Starck (1979) reminds us that these relationships not only show group specific differences, but also ontogenetic and possibly Fig. 1. Skull of Dinilysia patagonica, MLP 79-II-27-1. Scale bar equals 10 mm. (A) Dorsal view, (B) ventral view, (C) right lateral view and (D) left lateral view. Abbreviations: aa, anterior ampulla; av, aortic vessels; bo, basioccipital; bot, basioccipital tubers; cbl, cerebellum; ie, inner ear; lic, left inner carotid; oc, occipital condyle; p, parietal; pb, posterior brain; pg, pituitary gland; pr, prootic; pt, pterigoid; q, quadrate; ric, right inner carotid; so, supraoccipital; V, trigeminal nerves; VI, abducens nerves; vc, V f, foramen of the trigeminal nerves; VII f, foramen of the facial nerves, vidian canal; vv, vascular vessels. 10 TRIVI ~NO ET AL. sexual ones. Wyneken (2007) suggests that the nervous system of reptiles is relatively simple in anatomical struc- ture yet allows greater functional diversity in species- specific behaviors and adaptation to diverse niches. According to Hopson (1979), the reptile brain does not fill the brain cavity and the extra neural elements occupy part of the intracranial space. This would means that the endocast shows the place previously occupied by the brain and intracranial space, providing only a super- ficial overview of the topography of the brain (Jerison, 1969; Hopson, 1979; Norman and Faiers, 1996; Larsson et al, 2000; Wyneken, 2007). Although several groups of reptiles present mostly ossified braincases, the brain still does not completly occupy the cranial cavity; thus endo- casts would neither be representative of the brain topog- raphy in these cases. In this sense, a well-developed subdural space is observed in marine turtles, Sphenodon and many lizards. Most Testudines and Crocodylia pre- sent a moderate subdural space. However, this space is very narrow in snakes and amphisbaenians (Starck, 1979). Because of this, Wyneken (2007) considers that endocasts of snakes would allow us to hope a good mor- phological copy of their brains. Recently, Olori (2010) studied a digitized endocast of an uropeltid and enunci- ated the hypothesis that the relationship between the cranium and the endocraneal cavity is similar to that of mammals, involving the possibility that endocraneal space is completely occupied by the brain. This condition could be a consequence of the high degree of fusion in the bones of the uropelid skull, thus determining a completely closed cranial cavity (Olori, 2010). For this paper, several skulls of Dipsadidae (Colubroi- dea) were dissected to test the hypothesis of Olori (2010) in other extant snakes, focusing on the disposition of the brain and subdural space in the cranial cavity. These snakes were collected after they died flattened by cars on the road. The heads of some of these snakes were sec- tioned in sagital plane and others in longitudinal plane (Table 1), to observe the disposition of brain regions, the cranial space occupying these regions, and the arrange- ment of the veins and cranial nerves. As Figures 3 and 4 shows, the cranial bones surround the tissue of the ner- vous system very closely, leaving a very small space between the bone and the nervous tissue for the blood vessels to pass through. Thus, the brain fills around 90% of the endocranial cavity in snakes, leaving a very narrow intracavitary space. Fossil specimens examined. An exceptionally well- preserved specimen of Dinilysia patagonica (MLP 79-II- 27-1) was used for the present study. The material was collected by Santiago Roth at the end of XIX century in Boca del Sapo, Neuqu�en city, at the confluence of the Neuqu�en and Limay Rivers, Neuqu�en province, Argen- tina. The specimen was recovered from rocks belonging to the Bajo de La Carpa Formation (Santonian), R�ıo Col- orado Subgroup, Neuqu�en Group. It is represented by the posterior portion of a well preserved skull including the corresponding fragment of a natural endocast Fig. 2. Skull of Dinilysia patagonica, MLP 79-II-27-1. Scale bar equals 10 mm. (A) Dorsal view, (B) ventral view, (C) ventroanterior view zoom, (D) lateral view, (E) ventroposterior view zoom. Abbreviations: av, aortic vessels; bo, basioccipital; bot, basioccipital tubers; cbl, cerebellum; ie, inner ear; lic, left inner carotid; oc, occipital condyle; p, parietal; pb, posterior brain; pg, pituitary gland;; pr, prootic; q, quadrate; ric, right inner carotid; s, stapes; so, supraoccipital; V f, foramen of the trigeminal nerve; VII f, foramen of the facial nerve; VI, abducens nerves; vv, vascular vessels. FIRST ENDOCRANIAL CAST OF FOSSIL SNAKE 11 exposed on the dorsal right side. The specimen was mechanically prepared through the extraction of the fol- lowing bones to have better endocast exposure: parietal, prootic, a supraoccipital segment, and a portion of the right otooccipital. The extraction of these bones exposed the sedimentary fillings that occupied the place of the soft tissues of the brain, nerves, blood vessels, and inner ear, forming the first natural endocranial cast of a snake. The described specimen was directly compared with the following specimens of Dinilysia patagonica: MLP 26–410 (holotype), MACN-PV RN 1013, and MACN-PV RN 1014. Living specimens examined. The fossil specimen was also compared with skulls of the extant Boa constrictor occidentalis (UNMdP-O 44), Salvator merianae (MLP-R 5969, MLP-R 6029), Broghammerus reticulatus (MLP-R 6030), and Bothrops alternatus (MLP-R 6031). Moreover, for comparative studies, latex endocasts (CNP-ME 146, CNP-ME 147, CNP-ME 148) were obtained from skulls of the extant snake species Boa constrictor occidentalis (UNMdP-O 50, UNMdP-O 47), TABLE 1. Extant dissected individuals for comparative purpose Specie Collection number Provenance Date of collection Colector Dissection mode Erythrolamprus poecilogyrus MLP –R 6462 Ruta provincial 11, La Plata- Magdalena 11/14/2015 Jorge Williams Section longitudinal to the main axis of the body Erythrolamprus poecilogyrus MLP –R 6463 Ruta provincial 11, La Plata- Magdalena 11/14/2015 Jorge Williams Section parallel to the main axis of the body Thamnodynastes strigatus MLP –R 6464 Ruta provincial 11, La Plata- Magdalena 11/14/2015 Jorge Williams Dorsal dissection and left lateral Lygophis anomalus MLP –R 6465 Ruta provincial 11, La Plata- Magdalena 11/20/2016 Laura Trivi~no Bone conservation, observation of cranial nerves Lygophis anomalus MLP –R 6466 Ruta provincial 11, La Plata- Magdalena 12/09/2016 Laura Trivi~no Bone conservation, observation of cranial nerves Philodryas patagoniensis MLP –R 6467 Ruta provincial 11, La Plata- Magdalena 11/20/2016 Laura Trivi~no Section longitudinal to the main axis of the body Philodryas patagoniensis MLP –R 6468 Ruta provincial 11, La Plata- Magdalena 11/20/2016 Laura Trivi~no Dorsal dissection and right lateral Xenodon dorbignyi MLP –R 6469 Ruta provincial 11, La Plata- Magdalena 12/09/2016 Laura Trivi~no Dorsal dissection and right lateral Fig. 3. Dissection of skull of Erythrolamprus poecilogyrus. Scale bar equals 10 mm. (A) Dorsal view, (B) left lateral view. Abbreviations: cbl, cer- ebellum; cer, cerebrum; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe. 12 TRIVI ~NO ET AL. and Hydrodynastes gigas (UNMdP-O 54). Table 1 details the dissected skulls of Dipsadidae used in this study to see the relationships among the brain and the braincase. The material was studied using a stereoscopic micro- scope and was photographed with digital cameras. Abbreviations. CNP-ME: Colecci�on de moldes endocra- neanos del Centro Nacional Patag�onico (CENPAT), Puerto Madryn, Argentina; MACN-PV RN: Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Secci�on Paleontolog�ıa de Vertebrados, Colecci�on R�ıo Negro, CABA, Argentina; MLP: Museo de La Plata, Divisi�on Paleontolog�ıa de Vertebrados, La Plata, Argentina; MLP-R: Museo de La Plata, secci�on Zoolog�ıa de Vertebrados, Divisi�on Herpetolog�ıa, Reptiles, La Plata, Argentina; UNMdP-O: Universidad Nacional de Mar del Plata, Colecci�on Herpetol�ogica, Secci�on Osteolog�ıa, Mar del Plata, Argentina. Systematic Paleontology REPTILIA Linnaeus, 1758 SQUAMATA Oppel, 1811 SERPENTES Linnaeus, 1758 DINILYSIA Smith-Woodward, 1901 DINILYSIA PATAGONICA Smith-Woodward, 1901 Figures (1 and 2), 5–9, and Figure 12 Referred specimen. MLP 79-II-27-1 posterior portion of an articulated skull including the endocast. Provenance. Boca del Sapo, Neuqu�en city Neuqu�en province, Argentina. Horizon. Neuqu�en group, R�ıo Colorado Subgroup, Bajo de la Carpa Formation (Santonian, upper Cretaceous). Description Braincase and Basicranium Among elements of the braincase, the parietal, prootic, supraoccipital and otooccipital bones are well preserved on the left side (Figs. 1 and 2). The parietal bone forms the roof as well as the lateral walls of the braincase, and makes up the anterior limit of the trigeminal foramen. Furthermore, the parietal surrounds the dorsal part of the inner ear and posterior brain. On the left wall of the braincase, behind the parietal bone, the prootic forms the posterior margin of the trigeminal foramen. Poste- rior to the trigeminal foramen there is a small foramen for the facial nerve. Both foramina are separately found, each one having a sole opening where nerves pass through. In the occipital region, the supraoccipital, otooccipital, and basioccipital bones form the dorsal and posterior portion of the braincase and basicranium. The supraocci- pital is located dorsally and posteriorly to the parietal, united by a zig-zag suture. This bone covers the poste- rior and dorsal extreme of the myelencephalon. The otooccipitals and the basioccipital constitute the ellipsoi- dal occipital condyle. A small part of the supraoccipital and otooccipitals forms the foramen magnum. In the otooccipital portion of the spinal canal, near the foramen magnum, there is a pair of foramina; the anterior is the exit of the vagus nerve and the posterior foramen is for the hypoglossal nerve. The metotic foramen opens exter- nally, near the neck of the occipital condyle and over the otooccipitals. The glossopharyngeal, vagus and hyplo- glossal nerves, as well as the jugular vein, exit the cra- nial cavity through this foramen. The basicranium is formed by the basioccipital and basiparasphenoid (Figs. 1 and 2). Ventrally, the Fig. 4. Dissection of skull of Thamnodynastes strigatus. Scale bar equals 10 mm. (A) Dorsal view, (B) left lateral view. Abbreviations: b: bones; cbl, cerebellum; cer, cerebrum; i ear, inner ear; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe. FIRST ENDOCRANIAL CAST OF FOSSIL SNAKE 13 basioccipital has a pair of basal tubercles anterior to the occipital condyle, which also appears in other specimens of Dinilysia. The floor of the basiparasphenoid is lost, thus, it is possible to see the elements that run within this bone. The cast shows the pituitary gland sur- rounded by a groove that corresponds to the Vidian Canal. The internal carotid and the dorsal branch of the facial nerve (palatine ramus or Vidian nerve) pass through the Vidian Canal. A portion of nerve VI and sev- eral blood vessels, which would have irrigated the base of the brain, are also recognized. Natural Endocranial Cast The natural endocast preserved in the specimen MLP 79-II-27-1 exhibits the posterior brain, that is, the hind- brain (cerebellum and medulla oblongata). The forebrain is exposed in ventral view; it is represented by the dien- cephalon with the ventrally extended pituitary gland. The brain is horizontal, without flexures between regions. The natural endocast also includes sedimentary fillings corresponding to some cranial nerves, the right ear (middle and inner), and the impressions related to venous and arterial craniocerebral circulatory elements (Figs. 1 and 2). Brain In ventral view, the pituitary gland cast is observed as an expansion of the diencephalon. The position of this gland is posterior, next to the output of the trigeminal nerve (Figs. 1 and 2). Vessels related to the ventral cir- culatory system of the skull are immediately behind the gland. The Abducens nerve (VI) surrounds the vessels, and external to this nerve are the natural casts of the Vidian Canal system. The internal carotids, emerging from their respective Vidian Canals, enter at the most anterior part of the pituitary gland. Fig. 5. Blood vessels. Scale bar equals 10 mm. (A) posterior view of the venous vessels, (B) medial view of the venous vessels, (C) arterial vessels, (D) arterial vessels, (E) medial view of the venous vessels, (F) posterior view of the venous vessels. Abbreviations: c, capillaries; cc, common crus; cv, cerebral vein; lic, left inner carotid; lv, longitudinal vein; mcv, median cerebral vein; pg, pituitary gland; sv, venous sinuses; ric, right inner carotid. 14 TRIVI ~NO ET AL. The hindbrain is observed in dorsal view; it is formed by two smaller regions, the metencephalon and the mye- lencephalon. The structures observed in the hindbrain of the endocast are the following: cerebellum, cranial nerves V, VI and VII, and medulla oblongata. The cerebellum and cranial nerves V, VI, and VII are observed forming part of the metencephalon. Next to the inner ear and branching outwards from the lateral wall of this region are the trigeminal nerve (V) and the facial nerve (VII). The root of the trigeminal nerve is bigger than the facial nerve and is located in front of it. The abducens nerve (VI) is noted in the floor of the meten- cephalon, posterior to the exit of the trigeminal nerve and it runs near the pituitary gland, internal to the Vidian canal. The medulla oblongata is observed in the ventral face of the myelencephalon, where nerves IX, X and XII exit. The cast of the cerebellum is located in the medial region of the parietal, in the area where this bone begins to taper laterally. The cerebellum consists of a simple structure (corpus cerebelli) separated into two hemi- spheres by an anteroposterior groove where the dorsal longitudinal venous sinus that drains the brain would have run (Figs. 1, 2, and 5). The natural endocast only preserves the correspond- ing filling of the trigeminal (V), abducens (VI) and facial (VII) nerves. The trigeminal nerve (V) originates in the metencephalon and exits the braincase through a large and single foramen anterior to the inner ear (displayed on the left side) (Figs. 1D and 2). This nerve innervates the muscles of the jaw and the eye regions. The trigemi- nal ganglion corresponding to this nerve [5gasser (gas- serian) or lunate] is located outside the braincase; it is a motor-sensitive element which is divided into four branches (Figs. 6A and 7). The abducens nerves (VI) exit from the floor of the metencephalon, posterior to the tri- geminal nerve (Figs. 2C and 6B) and runs parallel to the pituitary gland in the ventral face of the basicranium. The facial nerves (VII) originate on the lateral wall of the metencephalon, posterior to the root of the trigemi- nal nerve (Figs. 6A and 7). They have a dorsal branch (hyomandibular branch) that goes lateroventrally to the external edge of the endocast immediately before the inner ear and the ventral branch (palatine branch), which is directed towards the posterior foramen of the Vidian canal and enters the channel. Externally, in the prootic bone, it is possible to see a single foramen that is independent from the trigeminal recess. Ear An area crossed by the massive columella (stapes) is located ventrolaterally, viewed from the occipital plane (Figs. 2E and 8A). The distal end of this element con- tacts the quadrate. Its major axis is directed towards the ventral part of the inner ear. The stapes is in contact with the inner ear through the footplate that rests in the oval window. The endocast of the right inner ear is preserved. It is a compact and massive structure located behind the base of the trigeminal nerve. The inner ear is predomi- nantly represented by a large element in the center, the vestibule, which would have contained a large central mass, the statolith, when the animal was alive (Figs. 3, 4, 7, 8B, and 9). This structure is ellipsoid shaped (although in lateral view it is spherical) and is compact in appearance. It is directed towards the midplane of the skull, passing over the medulla oblongata and behind the small lobes of the cerebellum. The vestibule is sur- rounded by very thin and close semicircular canals: Fig. 6. Natural endocranial cast. Scale bar equals 10 mm. (A) right lateral view, (B) ventral view. Abbreviations: aa, anterior ampulla; cbl, cerebellum; lsc, lateral semicircular canal; pb, posterior brain; pg, pitu- itary gland; pt, pterigoid; q, quadrate; V, trigeminal nerves; VI, abdu- cens nerves; VII h, hyomandibular facial nerves; VII p, palatine facial nerves; vb, vestibule; vc, vidian canal. Fig. 7. Diagram lateral view of natural endocranial cast. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semi- circular canal; cbl, cerebellum; pb, posterior brain; pg, pituitary gland; pt, pterigoid; q, quadrate; V, trigeminal nerves; VII h, hyomandibular facial nerves; VII p, palatine facial nerves; vb, vestibule. FIRST ENDOCRANIAL CAST OF FOSSIL SNAKE 15 anterior, posterior and lateral. These delicate canals maintain their continuous diameter and are smoothly curved around the vestibule. The lateral canal is long and positioned in a horizontal plane close to the vesti- bule and the quadrate. Between the lateral semicircular canal and the vestibule there is no bone separates them and the two structures are very close to each other. Also, the lateral canal follows the contour of the vestibule. The anterior and posterior semicircular canals originate from the foremost and hindmost ends of the vestibule, respectively. They are directed vertically and laterally to the median plane of the skull. In dorsal view, these canals form a flattened cone with a hemispherical end at the base, around the vestibule. The anterior and poste- rior canals form the sides of the cone, whereas the lat- eral canal produces the contour of the hemisphere. The anterior ampulla is also preserved, connecting the ante- rior canal and the small fragment of the lateral canal. The ampulla is a compact structure with an anterior prominence; it is preserved at the front end of the vesti- bule, very close to the exit of the trigeminal nerve. At the junction between the anterior and posterior semicir- cular canals, a very small common crus is observed. The semicircular canals are connected together forming dif- ferent angles: the angle is greater than 908 between the anterior and posterior canals, whereas it is close to 908 between the posterior and lateral canals, as well as between the anterior and lateral canals. Venous and Arterial Craniocerebral Circulation In the endocast of Dinilysia, the casts of the venous vessels run along the dorsal surface of the brain (Fig. 5). These vessels runs along the midline of the skull, and the branches in the posterior region are transformed into the posterior cerebral veins that exit the braincase through the jugular foramen. From the inner ear exit vessels that drain the vestibule (these vessels are thin and branched, and appear on the surface of the vesti- bule), and in the rostral end of the cerebellum they join the dorsal longitudinal venous sinus. On the ventral part of the cast, the mark left by the Vidian canal is observed with the corresponding refill of the internal carotid artery (which forms the cerebral carotid artery when it enters the brain) (Fig. 5). This artery reaches the front end of the pituitary gland, entering and irrigating the brain from the ventral region. The left carotid artery has a slightly larger diam- eter than the right one. Three thin unidentified vessels are observed behind the pituitary gland, the middle one enters the gland whereas the other two surround it laterally. DISCUSSION The material studied here is the first record of a natu- ral endocranial cast of an extinct snake. Studies on the central nervous system of fossil snakes are nonexistent whereas literature on this issue in recent snakes is scarce. The earliest work on the nervous system of recent snakes dates from the late nineteenth century by Edinger (1896), who made descriptions of fibers of the telencephalon of reptiles and included images of fiber tracts and cell masses of the forebrain of pythons. Auen and Langebartel (1977) described for the first time all cranial nerves in two species of colubrids (Elaphe obso- lete quadrivittata and Thamnophis ordinoides), identify- ing eleven cranial nerves. The spinal accessory nerve (XI) is absent in snakes. The trigeminal nerve contains four-branches. Branch V1 is a sensory nerve and reaches the jaw’s anterior end. Branch V4, like branches V2 and V3, has a motor component. This pterygoid branch (V4) is small to branched and innervates five muscles of the upper jaw. The output of the cranial nerves is evident in Fig. 8. (A) Ventral view of the middle ear. (B) Inner ear. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; bo, basioccipital; cc, common crus; lsc, lateral semicircular canal; oc, occipital condole; psc, posterior semicircular canal; s, stapes; vb, vestibule. 16 TRIVI ~NO ET AL. the arrangement of the skull bones of all reptiles, allow- ing identification both in existing and extinct taxa (Romer, 1956; Breazile, 1979; Saveliev, 2008; Carabajal, 2009). Rieppel and Zaher (2000) have noted in recent snakes that the two branches of the facial nerve usually exit the braincase through two separate foramina, except in those cases where the palatine branch pursues an intracranial course into the Vidian canal. In the stud- ied specimen of Dinilysia patagonica, one foramen is observed for the facial nerve, which is consistent with the condition observed in other skulls of this snake (Estes et al, 1970; Zaher and Scanferla, 2012). The brain surface of reptiles is typically smooth in dorsal view, presenting different structures that are placed consecutively one after the other. They are: the Forebrain, associated with the sensory organs develop- ing in the nasal capsule, and related to smelling and sensory-motor integration; the Midbrain, associated with the optical capsule, which carries out visual processing as well as neuroendocrine roles; and the Hindbrain, associated with the otic capsule, which carries out the role of hearing and balance, and also makes role of homeostasis (Wyneken, 2007). The dorsal view of the specimen MLP 79-II-27-1 studied in this paper presents the Hindbrain, which consists of two smaller regions: the metencephalon and the myelencephalon. The meten- cephalon contains the cerebellum dorsally; this has a very simple structure formed by two longitudinal lobes. Ventral to the cerebellum are the pons, the medulla oblongata, and the fourth ventricle. The trigeminal (V), abducens (VI), and facial (VII) nerves exit from the medulla. The vestibulocochlear nerve (VIII) is not observed in this material. The myelencephalon is formed by the medulla oblongata, and is the source of the remaining posterior cranial nerves, i.e., the glossophar- yngeal (IX), vagus (X), spinal accessory (XI) (nerve not present in snakes), and the hypoglossal (XII). In the endocast of Dinilysia patagonica the pituitary gland, which is a ventral projection of the diencephalon (poste- rior region of the forebrain) (Butler and Hodos, 2005; Wyneken, 2007), is located near the exit of the trigemi- nal nerve. It is interpreted as the ventral projection of the gland that is in contact with the base of the hindbrain. According to Wyneken (2007) the shape and size of both the brain and the sense organs are possibly related to the lifestyle of the snakes. Olori (2010) reconstructed the endocast of a recent fossorial snake (Uropeltis wood- masoni), which is simple in form, with little differentia- tion between the three main areas (Forebrain, Midbrain and Hindbrain). During the observation of latex endo- casts and the sections of skulls of recent semiaquatic and terrestrial colubrids and boids (Figs. 3, 4, 11, and Fig. 9. Diagram Inner ear. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; cbl, cerebellum; cc, common crus; lsc, lateral semicircular canal; pb, posterior brain; psc, posterior semicircular canal; q, quadrate; vb, vestibule. Fig. 10. Latex endocast, Hydrodynastes gigas. Scale bar equals 10 mm. (A) Dorsal view, (B) ventral view. Abbreviations: cbl, cerebel- lum; cer, cerebrum; i ear, inner ear; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe; opt ch, optic chiasma; pit gl, pituitary gland; II, optic nerves; V, trigeminal nerves; X, vagus nerves. FIRST ENDOCRANIAL CAST OF FOSSIL SNAKE 17 12), it was possible to recognize marked flexures between the metencephalon and myelencephalon (pon- tine flexure, where the myelencephalon is exposed slightly towards a more dorsal position), as well as between the midbrain and the forebrain (cephalic flex- ure, where the forebrain is exposed towards a dorsal position). Also, in dorsal view, there is a marked differ- entiation between areas, where the lobes of the cere- brum (telencephalon) are larger in comparison with the optic lobes (Figs. 3, 4, 11, and 12). In the natural endo- cast of Dinilysia the morphology is very similar to what Olori (2010) observed in her studies on Uropeltis. That is, the brain does not present strong differentiation between the size of the lobes, and in lateral view, it is developed in horizontal plane, without flexures. Taking into account that these two snakes are placed in a basal position in recent phylogenetic analyses (Hsiang et al., 2015), this similarity could be related to the primitive condition of the nervous system in both Uropeltis and Dinilysia According to Yi (2013, 2015) and Yi and Norell (2015), the degree of vestibular expansion in the inner ear is related to the lifestyle of snakes. These authors analyzed the inner ear of 34 species of modern and fossil snakes, and 10 species of lizards and amphisbaenians. The sam- ple included modern snakes in three habit groups: aquatic, terrestrial generalists, and burrowing. Accord- ing to Yi and Norell (2015), the burrowing species show a lateral semicircular canal partly fused with the vesti- bule; in contrast, aquatic species show an expanded dis- tance between the lateral semicircular canal and the vestibule. Nonburrowing terrestrial snakes display an intermediate state between these two types. Analyzing the virtual endocast of the extinct snake Dinilysia pata- gonica, Yi and Norell (2015) concluded that this snake shares with modern burrowing squamates a large spher- ical vestibule, a large foramen ovale, and slender semi- circular canals in the inner ear. The vestibule occupies most of the space defined by the three semicircular canals and contacts the lateral canal (Yi and Norell, 2015). Because this morphology only appears in squa- mates that actively burrow underground, Yi and Norell (2015) conclude that Dinilysia has a burrowing lifestyle. The present study of the natural cranial endocast of Dinilysia patagonica and the inner ear allow us to Fig. 11. Latex endocast, Boa constrictor occidentalis. Scale bar equals 10 mm. (A) Dorsal view, (B) ventral view. Abbreviations: cbl, cerebellum; cer, cerebrum; i ear, inner ear; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe; pit gl, pituitary gland; II, optic nerves; III, oculomotor nerves; V, trigeminal nerves; VII, facial nerves. Fig. 12. Reconstruction of Dinilysia patagonica. 18 TRIVI ~NO ET AL. confirm that this snake has an expanded and ellipsoid vestibule almost entirely occupying the space enclosed by the semicircular canals. The lateral canal is very close to the vestibule, there is not bone between them, and these elements are not fused together. This mor- phology coincides with that described by Yi and Norell (2015) for burrowing snakes on the basis of the 3-D reconstruction of the specimen MACN-RN 1014. Unfortunately, Yi and Norell (2015) overlooked the previous suggestion of a possible semifossorial mode of life for this snake given by Albino and Caldwell (2003). That is, the possibility that Dinilysia was a partly surface-active snake, which spent a part of its time below a nonconsolidate ground excavated by itself. Extreme fossorial habits (active burrowers that live underground most of their time, as scolecophidians, uro- peltids, anilioids, and amphisbaenians) are accompanied by relevant body features completely absent in Dinilysia patagonica, such as small size, reduction and lateraliza- tion of the eyes, and vertebrae with reduced or absent neural spines on depressed neural arches (Albino and Caldwell, 2003). In contrast, Dinilysia is characterized by a medium to large body size (more than 1.50 m according to Albino and Caldwell, 2003; exceeding 1.80 m according to Yi and Norell, 2015), large and dor- sally exposed orbits, and neural spines of vertebrae rela- tively high in depressed neural arches; all characteristics compatible with both semifossorial or semiaquatic lifestyles (Albino and Caldwell, 2003). Besides, the skull of Dinilysia is large (around 10 cm long), with an expanded posterior region, whereas the anterior region seems to be more delicate due to a proba- ble loosely connected joint between the premaxilla and maxilla. This condition would not be compatible with an active excavation of nearly compact ground with the skull, as is the case of true fossorial snakes that have small skulls with a consolidate and rigid snout (Cundall and Rossman, 1993). Nevertheless, the widened poste- rior region of the skull of Dinilysia would have been probably associated with a little neck delineation, pro- viding a good adaptation to move unconsolidated ground easily. Thus, the very large inner ear, with the lateral semicircular canal close to the vestibule as in burrowing snakes could be explained considering a semifossorial lifestyle for this snake. The stapes of Dinilysia patagonica is large and mas- sive, and connects the quadrate with the oval window, making possible transmission of environment vibration to the inner ear through it. This characteristic was also observed by Frazzetta (1999) in Xenopeltis unicolor, where the stapes has a foot plate of large diameter, which is greater than the total length of the bone. In addition, X. unicolor is a medium-sized snake that grows up to 1 m (Greene, 2000), and has a relatively robust body, a posteriorly wide braincase, with short and trian- gular quadrates, and relatively large orbits in dorsal position (Frazzetta, 1999), all characteristics also found in D. patagonica. Taking into account that X. unicolor is one of the largest semifossorial extant snake; thus, D. patagonica could have developed similar habits with a partly fossorial lifestyle. In conclusion, the characteristics found in the skull, vertebrae and natural endocast of Dinilysia patagonica as a whole support that this snake had a semifossorial mode of life. It is possible that these snakes maintained their voluminous bodies resting under unconsolidated sediment, leaving their large dorsal eyes exposed on the outside and feeling the ground vibrations through their enormous inner ear waiting to hunt any possible prey (Fig. 10). The basal position of Dinilysia patagonica in most phylogenetic analyses contradicts with the widespread view that all primitive snakes were small, burrowing forms that are gape-limited and eat small invertebrate prey (Gauthier et al., 2012; Hsiang et al., 2015; Yi and Norell, 2015). The alternative phylogenetic positions of Dinilysia (Zaher, 1998; Longrich et al., 2012; Reeder et al., 2015) demonstrate that large semifossorial snakes, which likely consumed prey of diverse shapes and sizes and spent part of its time below a nonconsolidate ground excavated by itself, would have appeared early in snake phylogeny (premacrostomatan) (Albino and Caldwell, 2003; Albino, 2007, 2011; Albino and Brizuela, 2014). This does not provide specific support to the presump- tion of a subterranean origin of snakes as opposed to the hypothesis of an aquatic origin for this group, but, together with the record of madtsoiids, indicate that the earliest diversification of terrestrial snakes (i.e., not strictly aquatic nor fully subterranean) probably occurred in Gondwana (Albino, 2011; Albino and Brizuela, 2014). ACKNOWLEDGMENTS We thank Marcelo Reguero, curator of the paleontologi- cal collection in MLP for the loan of the fossil specimens. The technician Pablo Puerta (Museo Paleontol�ogico Egi- dio Feruglio, Trelew, Argentina) prepared the skull of Dinilysia patagonica and obtained the latex casts. Leo- nel Acosta (MLP) completed a more exquisite prepara- tion of the fossil specimen. Celeste Scattolini, Carlos Santamaria-Mart�ın and Rebecca Doyle helped with the English version of the manuscript. Martina Charnelli drew the illustrations. To the reviewers Michael Cald- well and Jennifer Olori who significantly improved the manuscript. This work was supported by CONICET. LITERATURE CITED Albino AM. 2007. Lepidosauromorpha. In: Gasparini Z, Salgado L, Coria RA, editors. Patagonian mesozoic reptiles. Indiana: Indiana University Press. p 87–115. Albino AM. 2011. Evolution of Squamata Reptiles in Patagonia based on the fossil record. Biol J Linn Soc 103:441–457. Albino AM, Brizuela S. 2014. An overview of the South American fossil squamates. The Anat Rec p 349–368. Albino AM, Caldwell MW. 2003. H�abitos de vida de la serpiente Cret�acica Dinilysia patagonica Woodward. Ameghiniana 40:407– 414. Auen EL, Langebartel DA. 1977. The cranial nerves of the colubrid snakes Elaphe and Thamnophis. J Morphol 154:205–222. Breazile J. 1979. Systema nervosum centrale. In: Baumel J, King A, Lucas A, Breazile J, Evans H, editors. Nomina anatomica avium, an annotated anatomical dictionary of birds. London: Aca- demic Press. p 417–472. Butler AB, Hodos W. 2005. Comparative vertebrate neuroanatomy: evolution and adaptation. New York: John Wiley & Sons. Caldwell MW. 1999. Squamate phylogeny and the relationships of snakes and mosasauroids. Zool J Linn Soc 125:115–147. Caldwell MW, Albino AM. 2002. Exceptionally preserved skeletons of the Cretaceous snake Dinilysia patagonica Smith Woodward, 1901. J Vert Paleontol 22:861–866. FIRST ENDOCRANIAL CAST OF FOSSIL SNAKE 19 Caldwell MW, Calvo J. 2008. Details of a new skull and articulated cervical column of Dinilysia patagonica Woodward, 1901. J Vert Paleontol 28:349–362. Cundall D, Rossman DA. 1993. Cephalic anatomy of the rare Indo- nesian snake Anomochilus weberi. Zool J Linn Soc 109:235–273. Edinger L. 1896. Untersuchungen €uber die vergleichende Anatomie des Gehirns. Neue Studien €Uber das Vorderhirn der Reptilien. Abh Sneckenb naturf Ges 19:313–386. Edinger T. 1975. Paleoneurology 1804–1966. An annotated bibliog- raphy. Adv Anat Embryol Cell Biol 49:1–258. Estes R, Frazzetta TH, Williams EE. 1970. Studies on the fossil snake Dinilysia patagonica Smith Woodward: Part I. Cranial mor- phology. Bull Mus Comp Zool Harv 140:25–74. Filippi LS, Garrido AC. 2012. Nuevo registro del g�enero Dinilysia (Squamata, Serpentes) para la formaci�on Anacleto (Campaniano inferior-medio), Rinc�on de Los Sauces, Neuqu�en, Argentina. Ame- ghiniana 49:132–136. Frazzetta TH. 1999. Adaptations and significance of the cranial feeding apparatus of the sunbeam snake (Xenopeltis unicolor): Part I. Anatomy of the skull. J Morphol 239:27–43. Gauthier JA, Kearney M, Maisano JA, Rieppel O, Behlke AD. 2012. Assembling the squamate tree of life: perspectives from the phe- notype and the fossil record. Bull Peabody Mus Natl Hist 53:3– 308. Hecht MK. 1982. The vertebral morphology of the cretaceous snake Dinilysia patagonica Woodward. N Jb Geol Pal€aont Mlt 1982: 523–532. Hopson JA. 1979. Paleoneurology. In: Gans, C, Northcutt RG, Ulin- ski P, editors, Biology of the Reptilia, v. 9, Neurology A. London: Academic Press. Hsiang AY, Field DJ, Webster TH, Behlke ADB, Davis MB, Racicot RA, Gauthier JA. 2015. The origin of snakes: revealing the ecol- ogy, behavior, and evolutionary history of early snakes using genomics, phenomics, and the fossil record. BMC Evol Biol 15:87. Jerison HJ. 1969. Brain evolution and dinosaur brains. Am Nat 103:575–588. Jerison HJ. 1973. Evolution of the brain and intelligence. New York: Academic Press. Larsson HCE, Sereno PC, Wilson JA. 2000. Forebrain enlargement among non avian theropod dinosaurs. J Vert Paleontol 20:615– 618. Lee MSY, Scanlon JD. 2002. Snake phylogeny based on osteology, soft anatomy and ecology. Biol Rev 77:333–401. Linnaeus C. 1758. Systema Naturae per Regna tria Naturae secun- dum Classes, Ordines, Genera, Species, cum Characteribus, Dif- ferentiis, Synonyms, Locus, volume 1. Laurenti Salvii, Stockholm, 824 pp. Longrich NR, Bhullar BAS, Gauthier JA. 2012. A transitional snake from the Late Cretaceous period of North America. Nature 488: 205–208. Macrini TE, Rowe T, Archer M. 2006. Description of a cranial endo- cast from a fossil platypus, Obdurodon dicksoni (Monotremata, Ornithorhynchidae), and the relevance of endocranial characters to monotreme monophyly. J Morphol 267:1000–1015. Norman DB, Faiers T. 1996. On the first partial skull of an ankylo- saurian dinosaur from the Lower Cretaceous of the Isle of Wight, southern England. Geol Mag 133:299–310. Olori JC. 2010. Digital endocasts of the cranial cavity and osseous labyrinth of the burrowing snake Uropeltis woodmasoni (Alethi- nophidia: Uropeltidae). Copeia 1:14–26. Oppel M. 1811. Die Ordnungen, Familien, und Gattungen der Rep- tilien als Prodrom einer Naturgeschichte derselben. M€unchen, Joseph Lindauer. p 87. Paulina Carabajal A. 2009. El neurocr�aneo de los dinosaurios Ther- opoda de la Argentina. Osteolog�ıa y sus implicancias filogen�eticas. Tesis Doctoral, Facultad de Ciencias Naturales y Museo, Univer- sidad Nacional de La Plata. p 540. Rage JC, Albino AM. 1989. Dinilysia patagonica (Reptilia, ser- pents): mat�eriel vert�ebral additionnel du Cr�etac�e sup�erieur d’Argentine. Etude compl�ementaire des vertèbres, variations intrasp�ecifiques et intracolumnaires. Neues Jahrb Geol Pal€aontol Monatsh 1989:433–447. Reeder TW, Townsend TM, Mulcahy DG, Noonan BP, Wood PL Jr., Sites JW Jr., Wiens JJ. 2015. Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected place- ments for fossil taxa. PLOS One 10:e0118199. Rieppel O, Zaher H. 2000. The braincases of mosasaurs and Vara- nus, and the relationships of snakes. Zool J Linn Soc 129:489– 514. Romer AS. 1956. Osteology of the Reptiles. Chicago, Univ. Chicago Press. p 772. Saveliev SV. 2008. Neurobiological approaches in vertebrate paleon- tology. Paleontol J 42:573–580. Scanferla CA, Canale JI. 2007. The youngest record of the Creta- ceous snake genus Dinilysia (Squamata, Serpentes). S AM J Her- petol 2:76–81. Scanlon JD, Lee MSY. 2000. The Pleistocene serpent Wonambi and the early evolution of snakes. Nature 403:416–420. Smith-Woodward A. 1901. On some extinct reptiles from Patagonia, of the genera Miolania, Dinilysia and Genyodectes. Proc Zool Soc Lond 70(2):169–184. Starck D. 1979, Cranio-cerebral Relations in recent reptiles. In: Gans C, Northcutt RG, Ulinski P, editors. Biology of the Reptilia, v. 9. Neurology A. London: Academic Press, p. 1–38. Tchernov E, Rieppel O, Zaher H, Polcyn MJ, Jacobs IJ. 2000. A new fossil snake with limbs. Science 287:2010–2012. Trivi~no LN, Albino AM. 2015. Hallazgo de restos de la serpiente Dinilysia patagonica Smith-Woodward 1901 en una nueva locali- dad del Santoniano de Patagonia, Argentina. Est Geol 71:e033. https://doi.org/10.3989/egeol.41858.347. Witmer LM, Ridgely RC, Dufeau DL, Semones MC. 2008. Using CT to peer into the past: 3D visualization of the brain and ear regions of birds, crocodiles, and non avian dinosaurs. In: Endo H, Frey R, editors. Anatomical imaging: towards a new morphology. Tokyo, Japan: Springer. p. 67–88. Wyneken J. 2007. Reptilian neurology: anatomy and function. Vet Clin N Am Exot Anim Pract 10:837–853. Yi H. 2013. Inner-ear morphology suggests burrowing behaviour in early snakes. 73rd Society of Vertebrate Paleontology Meeting. Los �Angeles, EE.UU. October, 242. Yi H. 2015. Habitat specialization of early snakes: insights from the Late Cretaceous Gondwanan species. Libro de res�umenes, V Con- greso Latinoamericano de Paleontolog�ıa de Vertebrados (Colonia de Sacramento), 3. Yi H, Norell MA. 2015. The burrowing origin of modern snakes. Sci Adv 1:e1500743. Zaher H. 1998. The phylogenetic position of Pachyrhachis within snakes (Squamata, Lepidosauria). J Vert Paleontol 18:1–3. Zaher H, Scanferla CA. 2012. The skull of the Upper Cretaceous snake Dinilysia patagonica Smith-Woodward, 1901, and its phylo- genetic position revisited. Zool J Linn Soc 164:194–238. 20 TRIVI ~NO ET AL. https://doi.org/10.3989/egeol.41858.347